Plant Cell, Tissue and Organ Culture

, Volume 94, Issue 3, pp 253–259 | Cite as

Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.)

  • Martina Beranová
  • Slavomír Rakouský
  • Zuzana Vávrová
  • Tomáš Skalický
Research Note
First Online:
Received:
Accepted:

Abstract

A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO4 solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.

Keywords

CaMV 35S promoter Expression pattern Fluorescence Green fluorescent protein marker SAAT 

Abbreviations

BAP

6-Benzylaminopurine

CaMV 35S

Cauliflower mosaic virus promoter

GFP

Green fluorescent protein

GUS

β-Glucuronidase

Kn

Kanamycin

SEM

Scanning electron microscopy

NAA

Naphthalene acetic acid

gfp

Green fluorescent protein gene

mgfp5-ER

Modified gene for Green fluorescent protein

nptII

Neomycin Phosphotransferase II gene

nos (promoter)

Nopaline Synthase promoter

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Notes

Acknowledgements

The authors are grateful for the financial support received from Ministry of Education, Youth and Sport of the Czech Republic (grants 1M06030, 1PO5ME800). Linguistic revision was kindly performed by John McAvoy.

References

  1. Ananthakrishnan G, Xia X, Amutha S, Singer S, Muruganantham M, Yablonsky S, Fischer E, Gaba V (2007) Ultrasonic treatment stimulates multiple shoot regeneration and explant enlargement in recalcitrant squash cotyledon explants in vitro. Plant Cell Rep 26:267–276. doi: 10.1007/s00299-006-0235-1 Google Scholar
  2. Dong JZ, McHughen A (1993) An improved procedure for production of transgenic flax plants using Agrobacterium tumefaciens. Plant Sci 88:61–71. doi: 10.1016/0168-9452(93)90110-L Google Scholar
  3. Flores Solís JI, Mlejnek P, Studená K, Procházka S (2007) Application of sonication-assisted Agrobacterium-mediated transformation in Chenopodium rubrum L. Plant Soil Environ 49:255–260Google Scholar
  4. Fullner KJ, Cano LJ, Nester EW (1996) Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273:1107–1109. doi: 10.1126/science.273.5278.1107 Google Scholar
  5. Holford P, Hernandez N, Newbury HJ (1992) Factors influencing the efficiency of T-DNA transfer during co-cultivation of Antirrhinum majus with Agrobacterium tumefaciens. Plant Cell Rep 11:196–199Google Scholar
  6. Horsch RB, Fry JE, Hoffman NL, Einchholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  7. Hraška M, Heřmanová V, Rakouský S, Čurn V (2008) Sample topography and position within plant body influence the detection of the intensity of green fluorescent protein (GFP) fluorescence in the leaves of transgenic tobacco plants. Plant Cell Rep (in press). doi: 10.1007/s00299-007-0431-7
  8. Jordan MC, McHughen A (1988) Glyphosate tolerant flax plants from Agrobacterium mediated gene transfer. Plant Cell Rep 7:281–284CrossRefGoogle Scholar
  9. Joersbo M, Brunstedt J (1990) Direct gene transfer to plant protoplasts by mild sonication. Plant Cell Rep 9:207–210. doi: 10.1007/BF00232181 Google Scholar
  10. Joersbo M, Brunstedt J (1992) Sonication: a new method for gene transfer to plants. Physiol Plant 85:230–234. doi: 10.1034/j.1399-3054.1992.850215.x Google Scholar
  11. Ling HQ, Binding H (1997) Transformation in protoplast cultures of Linum usitatissimum and L. suffruticosum mediated with PEG and with Agrobacterium tumefaciens. J Plant Physiol 151:479–488Google Scholar
  12. Millam S, Obert B, Preťová A (2005) Plant cell and biotechnology studies in Linum usitatissimum – a review. Plant Cell Tissue Organ Cult 82:93–103. doi: 10.1007/s11240-004-6961-6 Google Scholar
  13. Mlynárová L, Bauer M, Nap JP, Preťová A (1994) High efficiency Agrobacterium-mediated gene transfer to flax. Plant Cell Rep. 13:282–285. doi: 10.1007/BF00233320 Google Scholar
  14. Rakouský S, Tejklová E, Wiesner I, Wiesnerová D, Kocábek T, Ondřej M (1999) Hygromycin B − an alternative in flax transformant selection. Biol Plant 42:361−369. doi: 10.1023/A:1002457000944
  15. Tejklová E (1992) Long-term in vitro shoot-tip culture and plant regeneration in flax. Rost. Výroba (Praha) 28:1009–1022 (In Czech)Google Scholar
  16. Trick HN, Finer JJ (1997) SAAT: sonicated-assisted Agrobacterium-mediated transformation. Transgenic Res 6:329–336. doi: 10.1023/A:1018470930944 Google Scholar
  17. Trick HN, Finer JJ (2000) Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-mediated transformation-treated soybean cotyledons. Lett Appl Microbiol 30:406–410CrossRefGoogle Scholar
  18. Wijayanto T, McHughen A (1999) Genetic transformation of Linum by particle bombardment. InVitro Cell Dev-Pl 35:456–465CrossRefGoogle Scholar
  19. Wróbel M, Zebrowski J, Szopa J (2004) Polyhydroxybutyrate synthesis in transgenic flax. J Biotechnol 107:41–54. doi: 10.1016/j.jbiotec.2003.10.005 Google Scholar
  20. Zaragozá C, Muñoz-Bertomeu J, Arrillaga I (2004) Regeneration of herbicide-tolerant black locust transgenic plants by SAAT. Plant Cell Rep 22:832–838. doi: 10.1007/s00299-004-0766-2 Google Scholar
  21. Zhan XC, Jones DA, Kerr A (1988) Regeneration of flax plants transformed by Agrobacterium rhizogenes. Plant Mol Biol 11:551–559. doi: 10.1007/BF00017455 Google Scholar
  22. Zhang LJ, Cheng LM, Xu N, Zhao NM, Li CG, Jing Y, Jia SR (1991) Efficient transformation of tobacco by ultrasonication. Biotechnology 9:996–997CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Martina Beranová
    • 1
  • Slavomír Rakouský
    • 1
    • 2
  • Zuzana Vávrová
    • 1
    • 2
  • Tomáš Skalický
    • 1
    • 2
  1. 1.Faculty of Health and Social StudiesUniversity of South BohemiaCeske BudejoviceCzech Republic
  2. 2.Faculty of Science, Department of GeneticsUniversity of South BohemiaCeske BudejoviceCzech Republic

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